JP7703030B2 - Genomic deletions in African swine fever vaccines allow efficient propagation in stable cell lines - Google Patents

Description

Field of invention

This disclosure provides details regarding the construction of a recombinant live attenuated African Swine Fever Virus (ASFV) vaccine for the prevention of ASF caused by various strains of ASFV, such as the highly pathogenic Georgia 2007 isolate ("ASFV-G"). The exemplary vaccine contains deletions that result in efficient growth in stable cell lines, allowing industrial-scale production of the ASFV vaccine.

background

African swine fever virus (ASFV) strain Georgia (ASFV-G) is the only member of the virus family Asfarviridae and is the causative agent of a pandemic disease currently affecting a large contiguous geographic area stretching from Central Europe to China and South Asia (Costard et al, Philos. Trans. R. Soc. London B Biol. Sci., (2009) 364:2683-96; O'Donnell et al, J. Virol., (2015a) 89:6048-56). This pandemic has caused significant economic losses, devastated the swine industry, and induced a global shortage of protein availability (O'Donnell et al, (2015a), supra). ASFV is very large and complex, with a 180-190 kilobase double-stranded DNA genome encoding more than 150 open reading frames (ORFs).

As no commercially available vaccine is available, disease control is based exclusively on controlling the geographic movement of susceptible animals and culling infected animals (Costard et al, supra). Effective experimental vaccines have been obtained based on the development of attenuated virus strains through genetic engineering of the pathogenic parent virus to delete specific viral genes related to viral pathogenesis (O'Donnell et al, (2015a), supra; O'Donnell et al, J. Virol., (2015b) 89:8556-66; O'Donnell et al, J. Virol., (2017) 91:e01760-16; Borca et al, J. Virol., (2020) 94:e02017-19). Understanding the function of viral genes in the viral pathogenic process in the natural host is a crucial step in the rational development of genetically engineered experimental vaccines. These experimental live attenuated vaccines constitute by far the more advanced approach towards developing effective measures to control ASF.

The main technical problem in developing a commercially viable ASF vaccine is that these virus strains only replicate efficiently in primary cultures of porcine macrophages, which hampers large-scale production at an industrial level. To address this issue, we herein disclose mutations that allow the propagation of ASFV vaccines in stable cell lines while retaining the ability to replicate in porcine macrophages and full attenuation when inoculated into domestic pigs.

In one embodiment, the present disclosure provides a genetically modified virus having a viral genome that is at least 95% identical to SEQ ID NO:1. In a specific embodiment, the viral genome is identical to SEQ ID NO:1.

Further provided herein is a vaccine composition against African swine fever virus (ASFV), comprising a genetically modified virus having a viral genome that is at least 95% identical to SEQ ID NO:1. In a particular embodiment, the ASFV is the ASFV-Georgia 2007 isolate (ASFV-G).

The disclosure further provides, in a further embodiment, a method for protecting pigs against ASFV comprising administering to the pig a live attenuated vaccine comprising a genetically modified virus having a viral genome that is at least 95% identical to SEQ ID NO:1 in an amount effective to protect said pig from clinical ASFV disease. In a particular embodiment, the ASFV is ASFV-G. In a particular embodiment, the amount effective to protect vaccinated pigs from clinical ASFV disease is a vaccine comprising ¹⁰² to ¹⁰⁶ _HAD50 of a genetically modified virus having a viral genome that is at least 95% identical to SEQ ID NO:1.

Further embodiments provided by the present disclosure are recombinant ASFV mutant viruses having a deletion or partial deletion of each of the ORFs MGF360-4L, MGF360-6L, X69R, MGF300-1L, MGF300-2R, MGF300-4L, MGF3608L, MGF360-9L, MGF360-10L, MGF360-11L. In certain embodiments, the virus comprises a deletion of a genome fragment that is at least 95% identical to SEQ ID NO:2. In some embodiments, the mutant ASFV is an ASFV-Georgia isolate. In some embodiments, the mutant ASFV has a genome that is at least 99% identical to SEQ ID NO:1. Such viruses may also be part of a vaccine composition against ASFV-G.

Further embodiments provided by the present disclosure are methods for protecting pigs against ASFV, comprising administering to the pig a live attenuated vaccine comprising a recombinant having a deletion or partial deletion of each of ORFs MGF360-4L, MGF360-6L, X69R, MGF300-1L, MGF300-2R, MGF300-4L, MGF3608L, MGF360-9L, MGF360-10L, MGF360-11L in an amount effective to protect the pig from clinical ASFV disease. In certain embodiments, the ASFV is ASFV-G. In some embodiments, the amount effective to protect an inoculated pig from clinical ASFV disease is a vaccine comprising ¹⁰² to ¹⁰⁶ _HAD50 of the genetically modified virus.

Further provided herein are embodiments of a method for producing ASFV in a cultured stable cell line at a titer of ^{104-107 HAD50} /mL _, the method comprising the steps of inoculating a cultured stable cell line with a genetically modified virus having a viral genome that is at least 95% identical to SEQ ID NO:1; incubating the inoculated cell line under conditions that allow for viral replication; and propagating the virus to a titer _of ^{104-107 HAD50} /mL. In some embodiments, the stable cell line is a porcine embryonic kidney cell line engineered to express bovine _αvβ6 integrin. In certain embodiments, the ASFV viral genome comprises a viral genome that is at least 99% identical to SEQ ID NO:1.

INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

The patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The novel features of the invention are set forth with particularity in the claims. The features and advantages of the invention are illustrated in the following detailed description and the accompanying drawings.

Figure 1 provides data presented in a graph showing the in vitro growth characteristics of ASFV-G-ΔI177L/ΔLVR (top), ASFV-G-ΔI177L/ΔLVR p7 (middle) and ASFV-G-ΔI177L/ΔLVR p11 (bottom) in stable porcine cell lines and primary porcine macrophage cultures. Cell cultures were infected with each of the viruses (MOI=0.01) and virus yields were titrated at the indicated post-infection time points. Data represent the average of results from three independent experiments. The virus detection sensitivity was ≥1.8 log ₁₀ HAD ₅₀ /ml. Significant differences (*) in virus yields between two viruses at a particular time point were determined using the Holm-Sidak method (α=0.05) without assuming a consistent standard deviation. All calculations were performed on the software GraphPad Prism version 8. TCID ₅₀ , 50% tissue culture infectious dose.

FIG. 2 provides a diagrammatic comparison of the genomes of the two viruses, ASFV-G-ΔI177LΔLVR and the parental ASFV-G-ΔI177L.

Figure 3 provides a schematic of the gene deletion approach to construct cell line adapted mutants of ASFV. ΔLVR, or partial deletions in ΔLVR that delete only MGF300 1L, 2R, 4L. The donor plasmid shown serves as a template for homologous recombination, a standard method to produce recombinant virus.

Highly pathogenic African swine fever virus (ASFV) strain Georgia (ASFV-G) is the causative agent of the current pandemic, including contiguous geographical areas from Central Europe to Asia and South Asia, causing enormous economic losses. There is no commercially available vaccine, and live attenuated strains developed from the pathogenic parent virus by genetic engineering are the most advanced experimental vaccines. Recently, the inventors developed a vaccine candidate by deleting the I177L gene from the genome of ASFV-G (see U.S. Patent Application No. 16/580,058). This mutant strain was shown to be highly safe and highly effective in inducing protection against ASFV-G challenge. A key technical limitation for translating this mutant (and other recombinant ASFV vaccines) into industrial production is that they only replicate in primary cultures of porcine macrophages. Herein, we present the development of a derivative strain (ASFV-G-ΔI177LΔLVR) containing a 10.8 kb deletion in the left variable region (LVR) that allows efficient growth in stable porcine cell lines (see U.S. Patent No. 9,121,010). The deletion in the LVR affects 10 viral genes at the left end of the genome. The deletion remained stable after more than 30 additional passages in continuous stable porcine cell lines. Importantly, ASFV-G-ΔI177LΔLVR maintained the same level of attenuation, immunological properties and protective efficacy against challenge with virulent ASFV-G as the parent vaccine. This is the first example showing that a rationally designed, highly effective ASF vaccine candidate can be grown in a stable cell line while maintaining genomic stability, enabling ASFV vaccine production at an industrial level.

Preferred embodiments of the present invention are shown and described herein. It will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Various alternatives to the embodiments of the invention described herein may be used in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents are covered by the invention.

Technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined. Reference is made herein to a variety of materials and methodologies known to those of ordinary skill in the art. Standard references illustrating the general principles of recombinant DNA technology include Sambrook et al., "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y., 1989; Kaufman et al., eds., "Handbook of Molecular and Cellular Methods in Biology and Medicine", CRC Press, Boca Raton, 1995; and McPherson, ed., "Directed Mutagenesis: A Practical Approach", IRL Press, Oxford, 1991.

In practicing the present invention, any suitable materials and/or methods known to one of skill in the art may be utilized. Materials and/or methods for practicing the present invention are described. Materials, reagents, etc. referred to in the following description and examples may be obtained from commercial sources unless otherwise indicated. The present invention describes methods and tools for producing genetically modified strains of ASFV.

As used in this specification and claims, the use of the singular forms "a," "an," and "the" includes plural referents unless the context clearly dictates otherwise.

As used herein, the terms isolated, purified, or biologically pure refer to a referenced material that is substantially or essentially free from components that normally accompany the material in its natural state.

The term "about" is defined as plus or minus ten percent of the stated value. For example, about 1.0 g means 0.9 g to 1.1 g and all values within that range, whether or not specifically stated.

The term "nucleic acid consisting essentially of" and grammatical variations thereof means a nucleic acid that differs from a reference nucleic acid sequence by no more than 20 nucleic acid residues and that performs the function of the reference nucleic acid sequence. Such variants include sequences that are shorter or longer than the reference nucleic acid sequence, have different residues at specific positions, or combinations thereof.

The term "adjuvant" refers to a substance or vehicle that nonspecifically enhances the immune response to an antigen. Adjuvants can include suspensions of minerals (alum, aluminum hydroxide, or aluminum phosphate) to which the antigen adsorbs; or water-in-oil emulsions in which a solution of the antigen is emulsified in mineral oil (e.g., Freund's incomplete adjuvant), sometimes including killed mycobacteria to further enhance antigenicity (Freund's complete adjuvant). Immunostimulatory oligonucleotides can also be used as adjuvants (see, e.g., U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants also include biological molecules, such as costimulatory molecules.

The term "administer"/"administration" refers to any method of providing a substance, such as a therapeutic agent, to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

The terms "coding sequence" and "coding region" as used herein refer to nucleotide and nucleic acid sequences, including both RNA and DNA, that code for the genetic information for the synthesis of RNA, protein, or any portion of RNA or protein.

The term "effective amount" of a composition provided herein refers to an amount of the composition capable of performing the particular function for which the effective amount is expressed. The exact amount required may vary from composition to composition and function to function, depending on recognized variables, such as the compositions and processes involved. An effective amount may be delivered in one or more applications. Thus, while it is not possible to specify an exact amount, an appropriate "effective amount" may be determined by one of ordinary skill in the art through routine experimentation.

The terms "I117L", "ASFV I117L", and "genome I117L" are synonymous and refer to the ASFV-G open reading frame I117L, which encodes a 177 amino acid protein and is located on the reverse strand at nucleotides 175473 to 176006 of SEQ ID NO:3.

In the context of the present invention, the term "cell line adaptive mutation" refers to a modification of the ASFV genome resulting in a complete or partial deletion of ORFs MGF360-4L, MGF360-6L, X69R, MGF300-1L, MGF300-2R, MGF300-4L, MGF3608L, MGF360-9L, MGF360-10L, MGF360-11L (Figure 2). In a particular embodiment, the term refers to a deletion of the ASFV-G genome sequence disclosed as SEQ ID NO:2.

In the context of the present invention, the term "non-functional genomic" MGF360-4L, MGF360-6L, X69R, MGF300-1L, MGF300-2R, MGF300-4L, MGF3608L, MGF360-9L, MGF360-10L, or MGF360-11L refers to modified genes or ORFs located in the genome of ASFV, where such modifications of the ASFV genes result in no gene product or in a gene product that is biologically non-functional compared to the unmodified functional ASFV genes. Such modifications may include, but are not limited to, complete or partial deletion of coding sequences, disruption of the open reading frame (e.g., insertion of a shift mutation or insertion of a nonsense codon), modification of upstream or downstream regulatory elements, and/or any other method currently known or conceivable that inactivates or knocks out the functional expression of such ASFV gene(s).

The term "ASFV-G-ΔI177LΔLVR" is used to describe a particular embodiment of the present disclosure, namely, the vaccine/virus having the genomic sequence of SEQ ID NO:1. The term "ASFV-G ΔI177L" is used to refer to the parental strain of "ASFV-G-ΔI177LΔLVR" but lacking the "cell line adaptive mutations."

The term "immunize" means to render a subject protected against an infectious disease, such as by vaccination.

For the purposes of the present invention, the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions (x100) with identical residues in two optimally aligned sequences divided by the number of positions being compared. Gaps, i.e., positions where a residue is present in one sequence in the alignment but not in the other, are considered to be positions with non-identical residues. Alignment of two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch, J Mol Biol, (1970) 48:3, 443-53). Computer-assisted sequence alignment may be suitably performed using standard software programs such as GAP, which is part of the Wisconsin Package version 10.1 (Genetics Computer Group, Madison, Wisconsin, USA), using a default scoring matrix with a gap creation penalty of 50 and a gap extension penalty of 3.

The phrases "high percent identical" or "high percent identity," and grammatical variations thereof, in the context of two polynucleotides or polypeptides, refer to two or more sequences or subsequences that have at least about 80% identity, at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide or amino acid identity when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm or by visual inspection. In one exemplary embodiment, the sequences are high percent identical over the entire length of the polynucleotide or polypeptide sequence.

The term "swine" generally refers to any member of the family Suidae, including domestic and wild pigs, hogs, and boars.

A "vaccine" is defined herein as a biological agent capable of providing a protective response in an animal to which it is delivered, but unable to cause serious disease. Vaccination results in immunity from disease. Thus, a vaccine stimulates antibody production or cell-mediated immunity against a disease-causing pathogen (e.g., ASFV). Immunity is defined herein as the induction of a significantly higher level of protection against lethality and clinical symptoms following vaccination in a pig population compared to a non-vaccinated group. In particular, the vaccines described in this invention protect a majority of vaccinated animals against the development of clinical symptoms and lethality of disease. The vaccines of this disclosure are typically genetically engineered (recombinant) mutant virus vaccines.

In the context of the present disclosure, the term "non-deficient in its replication" refers to a non-naturally occurring recombinant ASFV that is capable of replicating and/or producing viral progeny in vitro and/or in vivo, although such replication and/or viral progeny production may occur at a reduced level compared to the unmodified parental strain. Thus, it may be the case that such an ASFV is non-deficient in its replication in vitro (e.g., in cell culture), but in vivo in a mammal, such an ASFV is at least partially impaired in its replication, e.g., resulting in replication and/or viral progeny production below the limits of detection.

As used herein, the term "minimum dose" or "minimum effective dose" refers to a dose that exhibits no or minimal toxicity to the recipient, yet still results in the production of a desired result (e.g., protective immunity).

Viruses/vaccines

Provided herein is a novel mutant ASFV-G virus (SEQ ID NO:1) that results from a recombinant deletion of a portion of the I117L gene of the parent ASFV-G genome (described in U.S. Patent Application No. 16/580,058) and a deletion of an approximately 9 kb genomic region. The genomic nucleotide sequence of the specific recombinant mutant ASFV-GΔI177L (SEQ ID NO:1) is described herein, which differs from the genomic nucleotide sequence encoding the parent ASFV-G.

The exemplary mutant strain of ASFV-G (SEQ ID NO:1) is representative of a class of recombinant vaccines in which cell line adapted mutations exist, including, without limitation, deletion mutants, nonsense mutants, insertion mutants, frameshift mutants, and other mutants that result in the deletion, non-functionality, and non-expression of various ORFs within the mutation (SEQ ID NO:2). One exemplary virus disclosed herein also has a non-functional I117L ORF, but other recombinant viruses contemplated include mutations in other ORFs, including the regulatory elements of these ORFs that result in non-expression or non-translation of the respective proteins. Such mutants may be in the ASFV-G wild-type genomic background (SEQ ID NO:3).

Modifications intended to eliminate functional expression of a target protein or to reduce expression or activity of a target protein may involve mutations in the DNA or gene encoding the target protein, including, but not limited to, deletions of all or part of the target gene, including the open reading frame of the target locus, transcriptional regulators, such as the promoter of the target locus, and any other regulatory nucleic acid sequences located 5' or 3' of the open reading frame, insertions of premature stop codons in the open reading frame, and insertions or deletions that shift the reading frame and lead to premature termination of translation. Any technique known to those skilled in the art may be used to achieve such deletion mutations. Reduced levels of a target protein or reduced activity of a target protein may also be achieved with point mutations or insertions in the DNA or gene encoding the target protein. Mutation, insertion, and deletion variants of the disclosed nucleotide sequences and genes may be readily prepared by methods well known to those skilled in the art. Techniques used to achieve reduced levels and/or reduced activity of a target protein may include CRISPR/Cas, TALEN, and Zn-finger nucleases. Creating mutations, insertions, and deletions that are functionally equivalent to those specifically disclosed herein is well within the skill of one of ordinary skill in the art trained in the art.

The approaches described herein used to generate deletion mutations in ASFV-G can be used in different isolates of ASFV, including naturally occurring and recombinant strains (e.g., isolates circulating in Asia, Europe, or Africa). These approaches can vary according to methodologies known in the art, including, but not limited to, the use of different selection markers that allow for selection of recombinant virus upon purification, such as fluorescent proteins, enzymes that can be used with chromogenic substrates, such as β-glucuronidase or β-galactosidase, and drug selection markers. These approaches can also be used to generate any mutation to any individual ORF within the larger mutations described herein. For example, any single ORF selected from MGF360-4L, MGF360-6L, X69R, MGF300-1L, MGF300-2R, MGF300-4L, MGF3608L, MGF360-9L, MGF360-10L, or MGF360-11L, and mutations to the regulatory elements regulating the expression and translation of these ORFs that result in a non-functional protein being produced by that ORF. For all uses herein, the nomenclature utilized for these ORFs will be recognized by those of skill in the art as utilizing standard names for these ASFV ORFs.

Mutations in other ASFV strains and genotypes are also encompassed by the present disclosure. ASFV strains that contain synthetic mutations in a nucleic acid sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2 are included in the present invention. ASFV strains that contain a whole genome with 95%, 96%, 97%, 98%, or 99% or higher identity to SEQ ID NO:1 are also included in the present invention.

The present disclosure further contemplates combinations of other recombinant mutations and cell line adaptive mutations. As such, it is not only wild-type viruses that may be modified as disclosed herein, but also strains containing non-naturally occurring mutations in other genes or genomic regions (see, e.g., U.S. Patent No. 9,814,771).

The present disclosure provides that such rationally designed live attenuated ASFV-G mutants can be incorporated into immunogenic compositions to produce effective vaccines to protect animals, such as pigs, from clinical ASF disease upon exposure to ASFV-G. Accordingly, one object of the present invention is to provide a method for protecting animals against ASFV-G by administering an effective amount of a rationally designed live attenuated vaccine. In another embodiment, the present disclosure provides a method for eliciting a protective immune response in animals, preferably Suidae (e.g., domestic pig (Sus scrofa domesticus), wild pig (Sus scrofa scrofa), warthog (Potamochoerus porcus), bushpig (Potamochoerus larvatus), giant forest hog (Hylochoerus meinertzhageni), and feral pig). Such methods will typically include administering to such animals one or more of the ASFV immunogenic compositions and vaccines described herein.

The immunogenically effective amount of the immunogenic compositions disclosed herein may vary based on a number of parameters. In general, however, an effective amount per dosage unit for intramuscular application may be from about 10 ² 50% hemadsorption dose ("HAD ₅₀ ") to 10 ⁶ HAD _50. In practicing the methodology of the present invention, one, two, or more dosage units may be utilized. Dosage units may be readily modified by one of skill in the art to accommodate a desired volume or mass. Regardless of dosage unit parameters, the immunogenic compositions disclosed herein may be administered in an amount effective to generate an immune response.

Dosage levels of the active ingredients in the vaccines disclosed herein can be varied by one of skill in the art to achieve the desired results in a subject or for each application. As such, the dosage level selected can depend on a variety of factors, including, but not limited to, dosage form, combination with other treatments, severity of pre-existing conditions, and the presence or absence of adjuvants. In preferred embodiments, a minimal dose of the immunogenic composition is administered. Determining the minimum dose is well within the capabilities of one of skill in the art.

Vaccines of the invention can be prepared by conventional methods used for commercially available live attenuated ASFV vaccines. In certain embodiments, a susceptible substrate is inoculated with an ASFV mutant disclosed herein and allowed to grow until the virus replicates to a desired titer, after which the ASFV-containing material is harvested. The harvested material can then be formulated into a vaccine preparation with immunogenic properties. Any substrate capable of supporting replication of the recombinant viruses provided herein can be used in the present invention, including primary cultures of porcine peripheral blood macrophages or blood from infected pigs.

Virus replication and production in stable cell lines

The vaccines provided herein containing the cell line adaptive mutations of the present disclosure replicate and grow at increased rates in stable cell lines compared to viruses lacking the mutations. ASFV-G-I117L could grow in porcine macrophages and increase titer by 4-5 logs (over 3-5 days), but was unable to grow in the stable cell lines tested. When the adaptive mutations were added to the vaccine, the vaccine was able to grow at a rate comparable to the parent vaccine in macrophages and increased vaccine titer by 4-5 logs (over 3-6 days).

Formulation and administration

The vaccines provided herein include one of the recombinant viruses as defined above in live form, and a pharma- ceutically acceptable carrier or diluent typically used in such compositions. Carriers include stabilizers, preservatives, and buffers. Suitable stabilizers include, for example, SPGA (sucrose, phosphate, glutamate, and albumin), carbohydrates (sorbitol, mannitol, starch, sucrose, dextran, glutamate, and glucose), proteins (milk powder, serum, albumin, casein, or degradation products thereof. Suitable buffers include, for example, alkali metal phosphates. Acceptable preservatives include, but are not limited to, thimerosal, merthiolate, and gentamicin. Diluents include water, aqueous buffers (e.g., buffered saline), alcohols, and polyols (e.g., glycerol).

In some cases, the vaccines of the present invention also contain or include one or more adjuvants, including any substance included in the immunogenic composition formulation that enhances the immune response in the recipient induced by the immunogenic composition. In some cases, such adjuvants may include proteins, other components included with the recombinant virus. Other adjuvants may be included as additional components of the immunogenic composition, including categories such as aluminum salts (alum), oil emulsions, saponins, immune stimulating complexes (ISCOMs), liposomes, microparticles, nonionic block copolymers, derivatized polysaccharides, cytokines, and a wide variety of bacterial derivatives. Any suitable adjuvant known in the art may be utilized in practicing the invention disclosed herein. Factors influencing the choice of adjuvant include the animal species, the particular pathogen, the antigen, the route of immunization, and the type of immunity required, and may be readily determined by one of skill in the art.

The immunogenic compositions of the present disclosure may also include a carrier in addition to the recombinant virus. The carrier utilized in implementing the immunogenic compositions provided herein may be any known in the art and may be a liquid, solid, semi-solid, or gel. The type of formulation may be modified depending on the route of antigen administration. Preferably, the carrier is non-toxic to the recipient. One of skill in the art can readily select such a carrier for application to a recipient animal, such as poultry.

The vaccines provided herein may be administered intramuscularly, subcutaneously, intranasally or by injection in an amount effective to protect animals against challenge with a pathogenic strain of ASFV. The vaccine may be administered orally, through direct oral inoculation, dosed in drinking water, or through a feed delivery system. The effective amount of the recombinant virus may vary according to parameters considered by one of skill in the art. The effective amount may be determined empirically, if necessary, by one of skill in the art following any known method or the guidance provided in the Examples herein.

Having generally described the invention, the invention will be better understood by reference to certain specific examples. The examples are included herein to further illustrate the invention and are not intended to limit the scope of the invention, which is defined by the claims.

Example 1

Cell cultures and viruses

Primary cultures of porcine macrophages were prepared from porcine blood as previously described (O'Donnell et al., (2015b), supra). Preparation of macrophage cultures in 96-well plates for virus titer determination was also performed as previously described (O'Donnell et al., (2015b), supra).

Porcine epithelial cells (PEC) are cell subclones derived after more than 60 passages from the parent LFPKαvβ6 cell line (US Pat. No. 9,121,010), a porcine _embryonic kidney cell line engineered to express bovine _αvβ6 integrin. Cell cultures were passaged in culture in DMEM medium (Life Technologies, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (HI-FBS; Thermo Fisher Scientific, Waltham, MA) and 1% antibiotic-antimycotic (Thermo Fisher Scientific) at 37°C and 5% _CO2 .

As described below, ASFV-G-ΔI177LΔLVR (SEQ ID NO: 1) was generated after serial passage of the live attenuated vaccine candidate strain ASFV-G-ΔI177L (U.S. Patent Application No. 16/580,058).

Comparative growth curves were performed between ASFV-G-ΔI177L (parental strain) and ASFV-G-ΔI177LΔLVR (SEQ ID NO: 1) in primary porcine macrophages and stable porcine cell cultures. Preformed monolayers were prepared in 24-well plates and infected at an MOI of 0.01 (based on a previously determined HAD ₅₀ in primary porcine macrophage cell cultures). After 1 h of adsorption at 37°C and 5% CO ₂ , the inoculum was removed and cells were rinsed twice with PBS. Monolayers were then rinsed with macrophage medium and incubated for 2, 24, 48, 72, and 96 h at 37°C and 5% CO ₂ . At the appropriate time points post-infection, cells were frozen at <-70°C and thawed lysates were used to determine titers by HAD ₅₀ /ml in primary porcine macrophage cell cultures. All samples were run simultaneously to avoid inter-assay variability.

Virus titration was performed on primary porcine macrophage cell cultures in 96-well plates. Virus dilution and incubation were performed using macrophage medium. Virus presence was assessed by hemadsorption (HA) and virus titers were calculated by the method of Reed and Muench (Reed & Muench, Am. J. Hygiene, (1938), 27:493-497). ASFV Georgia (ASFV-G) used in animal challenge experiments is a field isolate kindly provided by Nino Vepkhvadze, Laboratory of the Ministry of Agriculture (LMA), Tbilisi, Republic of Georgia. ASFV DNA was extracted from infected cells and quantified as previously described. Full-length sequences of the viral genome were determined using an Illumina NextSeq 500 sequencer as previously described (Borca et al, Sci. Rep., (2018), 8:3154).

Example 2

Cell line mutations and adaptation.

ASFV-G-ΔI177L did not significantly replicate in more than 30 cell lines tested in our laboratory, including commercially available cell lines of porcine origin. In the majority of cases, only the first replication step was observed, as evidenced by the presence of intracellular expression of fluorescence due to expression of recombinant RFP present in the ASFV-G-ΔI177L genome, and the virus yields detected were either minor or nonexistent (data not shown).

ASFV-G-ΔI177L showed obvious cytopathic effect after six serial passages in stable porcine cell line cells. The first passage of ASFV-G-ΔI177L was performed using an MOI of 10. By the sixth passage, the ASFV-G-ΔI177L yield reached a titer of approximately 10 ⁵ HAD ₅₀ /ml. Further serial passages led to a virus yield of approximately 10 ⁷ HAD ₅₀ /ml (Figure 1).

The evaluation of genomic changes associated with the passage of ASFV-G-ΔI177L (ASFV-G-ΔI177LΔLVR) was performed in the virus obtained after six passages by next-generation sequencing (NGS). Compared to the parent virus, a deletion occurred between positions 16818 and 27660 of the viral genome, resulting in a deletion of 10842 bp. This genomic modification completely deletes the following genes: MGF360-6L, X69R, MGF300-1L, MGF300-1L, MGF300-2R, MGF300-4L, MGF360-8L, MGF360-9L, and MGF360-10L. In addition, the genomic modification also causes the deletion of the N-terminal part of the MGF 360-4L gene and the C-terminal part of the MGF360-11L gene (Figure 2). This deletion results in the production of a novel hybrid protein, MGF360-41/11L. The resulting ORF, on the reverse coding strand, has 839 nt of MGF-360-11L combined with 592 nt of MGF-360-4L. The resulting ORF is composed of 1432 nt that encodes a novel 476 amino acid protein (SEQ ID NO:4).

The genomic stability of ASFV-G-ΔI177LΔLVR was further evaluated in virus populations obtained after 20 and 30 passages. Surprisingly, NGS analysis showed that there were no major further genomic changes when compared to the genome of the virus obtained after 6 passages. This result indicates that the viral genome of ASFV-G-ΔI177LΔLVR remains stable upon serial passages in stable porcine cell line cells.

Replication of ASFV-G-ΔI177LΔLVR in primary porcine macrophages

The goal of developing aptASFV-G-ΔI177L is to use it as a vaccine strain. Therefore, it is important to evaluate its ability to replicate in porcine macrophages, the main cells targeted by ASFV during infection in pigs. The in vitro growth characteristics of ASFV-G-ΔI177LΔLVR were then evaluated in primary porcine macrophage cells in a multi-step growth curve. Two different passages of ASFV-G-ΔI177LΔLVR in PEC were tested: the 6th and 12th passages. Cell cultures were infected at a multiplicity of infection (MOI) of 0.01 and samples were collected 2 h postinfection and every 24 h for the following 6–8 days. The results showed that both ASFV-G-ΔI177LΔLVR strains displayed similar growth kinetics compared to that of the parent ASFV-G-ΔI177L (Figure 1). Thus, the ability of ASFV-G-ΔI177LΔLVR to grow in stable porcine cell line cells does not affect its ability to replicate in vitro in primary porcine macrophage cell cultures.

Example 3

Animal testing

Animal studies were performed in the Plum Island Animal Disease Center (PIADC) animal facility under Biosafety Level 3-Agriculture (3-AG) conditions in accordance with a protocol approved by the USDA/DHS PIADC Institutional Animal Care and Use Committee (Protocol No. 225.04-16-R, 09-07-16).

To assess whether the genomic changes in ASFV-G-ΔI177LΔLVR caused any modification in the attenuated phenotype of the parent virus ASFV-G-ΔI177L, a group of 80-90 lb pigs was inoculated intramuscularly (IM) with a high dose of 10 ⁶ hemadsorption doses (HAD ₅₀ ) and the animals were observed for clinical evolution over a period of 28 days. None of the five inoculated animals showed any ASF-related signs and remained clinically normal during the entire observation period, indicating that ASFV-G-ΔI177LΔLVR remains fully attenuated (Table 1). Thus, ASFV-G-ΔI177LΔLVR maintains the fully attenuated phenotype of the parent virus.
Table 1. Pig survival and fever response after infection with different doses of ASFV-G-ΔI177LΔLVR.

Protective efficacy of ASFV-G-ΔI177L against exposure to parent ASFV-G

To evaluate the ability of ASFV-G-ΔI177LΔLVR infection to induce protection against challenge with the highly pathogenic parent virus ASFV-G, all animals infected with ASFV-G-ΔI177LΔLVR were challenged 28 days later with ¹⁰² _HAD50 of ASFV-G by the IM route. An additional group of five naive animals was challenged as mock-inoculated controls. All mock-inoculated animals began to show disease-associated signs by 3-4 days post-challenge (dpc), with a rapid increase in disease severity over the following hours; animals were euthanized at approximately 5 dpc (Table 2). On the other hand, the group of animals infected with ASFV-G-ΔI177LΔLVR remained clinically healthy and did not show any significant signs of disease during the 21-day observation period. Thus, ASFV-G-ΔI177LΔLVR treated animals are protected against clinical disease upon challenge with the highly pathogenic parental virus.

To quantify the efficacy of ASFV-G-ΔI177LΔLVR in protecting pigs against challenge with parental pathogenic ASFV-G, three groups of pigs were inoculated IM with either 10 ² , 10 ⁴ , or 10 ⁶ HAD ₅₀ ASFV-G-ΔI177LΔLVR, respectively. All animals remained clinically normal for a period of 28 days before being challenged IM with 10 ² HAD ₅₀ parental pathogenic ASFV-G. Animals were observed for 21 days. All animals remained clinically normal after challenge without showing any ASF clinically relevant signs (Table 2). Thus, ASFV-G-ΔI177LΔLVR efficacy is comparable to that reported for ASFV-G-ΔI177L (U.S. Patent Application Serial No. 16/580,058).
Table 2. Pig survival and fever response in ASFV-G-ΔI177LΔLVR infected animals exposed to the parental ASFV-G virus.

Example 4

Recombinant virus construction

Recombinant ASFVs lacking one or more genes have been produced in our and other laboratories. A donor plasmid is created that contains flanking arms of identical DNA (typically 1000 bp) on either side of the region targeted for deletion, and between these two arms is a reporter cassette (i.e. GFP, RFP) that allows for the selection of recombinant ASFVs. Introduction of this plasmid into virus-infected cells allows exchange of the donor plasmid for ASFV DNA. This occurs specifically in the homologous DNA arms. Examples of this are the deletion of individual genes I177L, 9GL, UK (O'Donnell et al, (2015b), supra; O'Donnell et al, (2017), supra; Borca et al; (2020), supra) or the deletion of multiple MGF genes (O'Donnell et al, (2015a), supra). FIG. 3 shows a schematic of how this can be done to create a precise deletion of the ΔLVR in the ASFV-G genome in 4A, and a partial deletion in the ΔLVR region that deletes only the three genes MGF3001L, 2R, 4. The same methodology can be applied to delete any gene or any group of genes in the cell line adaptation mutation region (SEQ ID NO:2), or other parts of the genome. Selection and screening of recombinant viruses can be performed as described herein or by any means known in the art.

Although the invention has been described with reference to details of exemplary embodiments, these details are not intended to limit the scope of the invention as defined in the appended claims. The embodiments of the present disclosure in which an exclusive property or privilege is claimed are defined in the following claims.

Claims (17)

A genetically modified virus, the genome of which comprises a viral genome having at least 95% identity to SEQ ID NO:1. The virus of claim 1, wherein the genome of the virus comprises SEQ ID NO:1. A vaccine composition against African swine fever virus (ASFV), comprising the genetically modified virus of claim 1. The vaccine composition of claim 3, wherein the ASFV is the ASFV-Georgia 2007 isolate (ASFV-G). A method for protecting a pig against ASFV, comprising administering to the pig an attenuated live vaccine comprising the genetically modified virus of claim 1 in an amount effective to protect the pig from clinical ASFV disease. The method of claim 5, wherein the ASFV is ASFV-G. 6. The method of claim 5, wherein the vaccine comprises 10 ² to 10 ⁶ HAD ₅₀ of the genetically modified virus of claim 1, in an amount effective to protect the pig from clinical ASFV disease. A recombinant ASFV mutant virus comprising a deletion or partial deletion of each of the following ORFs: MGF360-4L, MGF360-6L, X69R, MGF300-1L, MGF300-2R, MGF300-4L, MGF3608L, MGF360-9L, MGF360-10L, MGF360-11L , wherein the virus comprises a deletion of a genomic fragment having at least 95% identity to SEQ ID NO:2 . The recombinant virus of claim 8, wherein the ASFV is the ASFV-Georgia isolate. The recombinant virus of claim 8, wherein the recombinant ASFV mutant virus comprises a genome having at least 99% identity to SEQ ID NO:1. A vaccine composition against ASFV-G, comprising the recombinant virus according to claim 8. A method for protecting a pig against ASFV, comprising administering to the pig an attenuated live vaccine comprising the recombinant virus of claim 8 in an amount effective to protect the pig from clinical ASFV disease. The method of claim 12 , wherein the ASFV is ASFV-G. 14. The method of claim 13 , wherein the vaccine comprises 10 ² to 10 ⁶ HAD ₅₀ of the genetically modified virus of claim 8, in an amount effective to protect the pig from clinical ASFV disease. A method for producing ASFV at a titer of ¹⁰⁴ to ¹⁰⁷ _HAD50 /mL in a cultured stable cell line, comprising the steps of inoculating the cultured stable cell line with the virus described in claim 1; incubating the inoculated cell line under conditions that allow viral replication; and propagating the virus to a titer of ¹⁰⁴ to ¹⁰⁷ _HAD50 /mL. The method of claim 15 , wherein the stable cell line is a porcine embryonic kidney cell line engineered to express bovine _αvβ6 integrin. The method of claim 15 , wherein the ASFV comprises a viral genome having at least 99% identity to SEQ ID NO:1.

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